Low-temperature-resistant heterotrophic nitrifying and aerobic denitrifying bacteria and method for wastewater denitrification

Abstract

A low-temperature-resistant heterotrophic nitrification and aerobic denitrification bacteria is used for wastewater denitrification method, relates to a kind of biological bacteria for wastewater denitrification method, the bacteria is Pseudomonas umsongensis BK17, belongs to Pseudomonas, is preserved in China typical culture preservation center, and the preservation number is CCTCC M 20242924, and the preservation date is December 27, 2024.The strain is a heterotrophic nitrification-aerobic denitrification bacteria, can efficiently remove inorganic nitrogen pollution in water body, and still has good denitrification performance under low temperature stress, and has strong low temperature tolerance.The strain can efficiently treat nitrogen-containing wastewater at 10 DEG C, and the removal efficiency can reach more than 99%.In the inorganic nitrogen pollution treatment and application of various water bodies such as domestic sewage in cold regions of China, it has great potential.

Description

Technical Field

[0001] This invention relates to environmental microorganisms and their methods for wastewater denitrification, specifically to a method for wastewater denitrification using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria. Background Technology

[0002] In recent years, although water pollution has improved, nitrogen discharge into water bodies due to human activities remains a serious problem. Therefore, removing nitrogen from wastewater remains a crucial issue in addressing water pollution. Current methods for water pollution control include physical, chemical, and biological methods, with biological methods being particularly effective in treating nutrients in wastewater. Traditional biological nitrogen removal methods generally fall into two categories: conventional biological methods rely on autotrophic nitrifying bacteria (AOB and NOB) to convert ammonia to nitrate in an aerobic environment, and heterotrophic nitrifying bacteria to reduce nitrate to gas through denitrification in an anaerobic / anoxic environment. Conventional bioecological nitrogen control technologies typically separate nitrification and denitrification into two stages to maintain suitable growth conditions for autotrophic and heterotrophic microorganisms. This requires separate processes in two reactors, which is wasteful of resources and is complex and inefficient. With the discovery of heterotrophic nitrifying aerobic denitrifying bacteria, biological nitrogen removal technology has taken a significant step forward. Heterotrophic nitrification-aerobic denitrification can remove ammonia, nitrates and organic matter simultaneously in a single process, and is currently the most promising technology.

[0003] Northern my country experiences long, cold winters, making it difficult to efficiently remove nitrogen nutrients from cold lakes using biological methods. Current biological wastewater denitrification technologies are ineffective at low temperatures and suffer from a lack of low-temperature bacterial resources. Research has shown that low-temperature environments (<15℃) inhibit the growth and metabolism of autotrophic nitrifying bacteria. To overcome this limitation, researchers have continuously innovated and improved their methods, isolating and screening cold-resistant denitrifying microorganisms. Heterotrophic nitrifying-aerobic denitrifying bacteria have gradually attracted widespread attention due to their strong adaptability to low-temperature environments. More and more cold-resistant heterotrophic nitrifying-aerobic denitrifying functional bacteria have been screened and isolated from various environments, and their low-temperature resistance has been gradually discovered, making them a feasible alternative for treating nitrogen-containing pollutants and improving denitrification efficiency in cold regions. Summary of the Invention

[0004] The purpose of this invention is to provide a method for wastewater denitrification using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria, which provides *Pseudomonas aeruginosa* (…). Pseudomonas umsongensis BK17 is highly efficient at treating nitrogen-containing wastewater at a low temperature of 10℃. This strain achieves rapid degradation of ammonia nitrogen and nitrate nitrogen within 48 hours, and has a significant effect on removing inorganic nitrogen pollution from water bodies under low temperature conditions.

[0005] This invention provides the following technical solution:

[0006] A method for denitrifying wastewater using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria is disclosed. The heterotrophic nitrifying aerobic denitrifying bacteria is *Pseudomonas cucurbita*, specifically *Pseudomonas yinchengensis* BK17, belonging to the genus *Pseudomonas*. The strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20242924 and a deposit date of December 27, 2024.

[0007] (1) Source of strain:

[0008] The bacterial strains screened in the experiment were obtained from water samples collected from activated sludge in the aerobic tanks of wastewater treatment plants.

[0009] (2) Culture medium formulation:

[0010] Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L;

[0011] Nitrification liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml;

[0012] Denitrification liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml;

[0013] Nitrified solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L;

[0014] Denitrification solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L;

[0015] Mixed culture medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.2675 g / L, KNO3 0.2164 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, and 2 ml of trace element solution;

[0016] (3) Specific process:

[0017] 1) Prepare enrichment medium. Add the sample to a 250ml Erlenmeyer flask containing 100mL of enrichment medium and incubate for 48h. Set the constant temperature shaker parameters to 10℃ and 120 r / min. When the cells grow to the point that the medium is obviously turbid, transfer 10mL of the cell suspension from the turbid medium to 100ml of fresh enrichment medium and continue to incubate at 10℃ and 120 r / min for 48h. Repeat the above process 3 times.

[0018] 2) After enrichment, the samples were diluted using a gradient dilution method to obtain bacterial suspensions of various gradients, such as 10⁻¹, 10⁻², ..., 10⁻⁸. Three gradients, 10⁻⁶, 10⁻⁷, and 10⁻⁸, were selected, and 100 μL of each gradient was evenly spread on heterotrophic nitrification solid medium. The suspensions were incubated in a constant temperature incubator at 10℃ for 2-3 days. Single colonies will form in the medium. Single colonies were picked and streaked multiple times in a three-zone heterotrophic nitrification solid medium to ensure the purity of the strains in the medium.

[0019] 3) The obtained pure strains were inoculated onto denitrification medium and incubated in a biochemical incubator at 10 ºC for 72 h; strains with denitrification ability were preliminarily screened based on the color change of the denitrification medium, and strains with blue halos were selected for further screening.

[0020] 4) The selected strains were inoculated into nitrification liquid medium and cultured with shaking at 10 ºC and 120 rpm. After 60 h, the ammonia nitrogen removal efficiency was measured, and the strain with the highest efficiency, BK17, was selected for subsequent experiments.

[0021] (4) Application of wastewater denitrification methods:

[0022] The process includes the following steps: Heterotrophic nitrifying aerobic denitrifying bacteria are cultured under different conditions at low temperatures, and their ammonia nitrogen removal efficiency is tested; the carbon sources for nitrogen-containing wastewater are sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate, with a C / N ratio of 4-20, a pH of 5-10, a rotation speed of 40-200 rpm, an inoculum size of 0.5%-4%, and a salinity of 0-4%.

[0023] The aforementioned method for denitrifying wastewater using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria includes the Pseudomonas BK17 strain which removes one or a combination of ammonia nitrogen and nitrate nitrogen under low-temperature conditions.

[0024] The technical advantages of this invention are:

[0025] 1. The present invention relates to a low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacterium, which is *Pseudomonas aeruginosa*, named *Pseudomonas aeruginosa* BK17, belonging to the genus *Pseudomonas*, and its preservation number is CCTCC M 20242924.

[0026] 2. The application of the strain of the present invention in the efficient treatment of nitrogen-containing wastewater at 10℃. Under the low temperature condition of 10℃, the strain can achieve rapid degradation of ammonia nitrogen and nitrate nitrogen within 48 h.

[0027] 3. The strain of this invention maintains good denitrification activity at low temperatures and can be used for wastewater denitrification treatment in geographical areas with large annual temperature differences in northern and northwestern my country.

[0028] 4. The application of the low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria of the present invention in the treatment of nitrogen-containing wastewater under different environmental conditions: when the C / N ratio of nitrogen-containing wastewater is 8-16, the pH value is 6-8, the rotation speed is 120-160 rpm, and the inoculum amount is 3%-5%, the rapid degradation of nitrogen-containing wastewater is achieved. Attached Figure Description

[0029] Figure 1 This is a phylogenetic tree of the heterotrophic nitrifying aerobic denitrifying bacterium BK17 of the present invention using 16S-rRNA;

[0030] Figure 2 A schematic diagram of the denitrification performance of BK17 at low temperature (10℃) with ammonia nitrogen as the sole nitrogen source;

[0031] Figure 3 This is a schematic diagram of the denitrification performance of BK17 at low temperature (10℃) with nitrate nitrogen as the sole nitrogen source;

[0032] Figure 4 This is a schematic diagram of the denitrification performance of BK17 at low temperature (10℃) using ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source;

[0033] Figure 5 The effects of different carbon sources on the growth and ammonia nitrogen removal rate of this heterotrophic nitrifying aerobic denitrifying strain;

[0034] Figure 6 The effects of different C / N ratios on the growth and ammonia nitrogen removal rate of this heterotrophic nitrifying aerobic denitrifying strain were investigated.

[0035] Figure 7 The effects of different pH values ​​on the growth and ammonia nitrogen removal rate of this heterotrophic nitrifying aerobic denitrifying strain;

[0036] Figure 8 The effects of different rotation speeds on the growth and ammonia nitrogen removal rate of this heterotrophic nitrifying aerobic denitrifying strain were investigated.

[0037] Figure 9 The effects of different inoculum amounts on the growth and ammonia nitrogen removal rate of this heterotrophic nitrifying aerobic denitrifying strain were investigated. Detailed Implementation

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific examples. The examples described herein are only for explaining and illustrating the present invention, but the scope of protection of the present invention is not limited to the following examples. Example 1

[0039] The present invention discloses a heterotrophic nitrifying aerobic denitrifying bacterium, which is *Pseudomonas cucurbita*, specifically *Pseudomonas yinchengensis* BK17, belonging to the genus *Pseudomonas*. The strain is deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, Hubei Province, with accession number CCTCC M 20242924 and deposit date of December 27, 2024.

[0040] (1) Source of strain:

[0041] The strains screened in the experiment were obtained from water samples collected from the activated sludge of the aerobic tank at Zhenxing Wastewater Treatment Plant in Shenyang City, Liaoning Province.

[0042] (2) Culture medium formulation:

[0043] Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L;

[0044] Nitrification liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml;

[0045] Denitrification liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml;

[0046] Nitrified solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L;

[0047] Denitrification solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L;

[0048] Mixed culture medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.2675 g / L, KNO3 0.2164 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, and 2 ml of trace element solution;

[0049] (3) Specific process:

[0050] 1. Prepare enrichment medium. Add the sample to a 250ml Erlenmeyer flask containing 100mL of enrichment medium and incubate for 48h. Set the constant temperature shaker parameters to 10℃ and 120 r / min. When the cells grow to the point that the medium becomes obviously turbid, transfer 10mL of the cell suspension from the turbid medium to 100ml of fresh enrichment medium and continue incubating at 10℃ and 120 r / min for 48h. Repeat the above process 3 times.

[0051] 2. After enrichment, the samples were diluted using a gradient dilution method to obtain bacterial suspensions of various gradients, such as 10⁻¹, 10⁻², ..., 10⁻⁸. Three gradients, 10⁻⁶, 10⁻⁷, and 10⁻⁸, were selected, and 100 μL of each was evenly spread on heterotrophic nitrification solid medium. The suspensions were then incubated at 10℃ for 2-3 days. Single colonies will form in the medium. These single colonies were picked and streaked multiple times in a three-zone streak culture medium to ensure the purity of the bacterial strains in the medium.

[0052] 3. The obtained pure strains were inoculated onto denitrification medium and incubated in a biochemical incubator at 10 ºC for 72 h. Based on the color change of the denitrification medium, strains with denitrification ability were preliminarily screened, and strains showing a blue halo were selected for further screening.

[0053] 4. The selected strains were inoculated into nitrification liquid medium and cultured with shaking at 10 ºC and 120 rpm. After 60 h, the ammonia nitrogen removal efficiency was measured, and the strain with the highest efficiency, BK17, was selected for subsequent experiments.

[0054] Example 2:

[0055] Strain identification:

[0056] The selected strains were inoculated into nitrification medium and cultured at 10℃ and 120 rpm for 60 h. The cultured bacterial solution was then sent to Shanghai Meiji Biomedical Technology Co., Ltd. Sequence analysis and homology comparison were performed online using the NCBI database. A phylogenetic tree was constructed based on the 16S rDNA gene sequence of this strain and some phylogenetically related strains. The results are as follows: Figure 1 .

[0057] Example 3:

[0058] Denitrification performance of Pseudomonas yinchengensis BK17 at low temperature (10℃) with ammonia nitrogen as the sole nitrogen source

[0059] The BK17 strain from Example 1 was inoculated into nitrified medium and then cultured in a shaker at 10°C. After it reached the logarithmic growth phase, 1% by volume of the culture solution was inoculated into 100 mL of NH4+. + The strain was cultured for 60 h in a nitrification medium with -N as the sole nitrogen source (concentration 100 mg / L) at 10 °C and 120 r / min. Samples were taken at 0, 12, 24, 36, 48, and 60 h, and the heterotrophic nitrification performance of the strain was tested after centrifugation and filtration. Simultaneously, NO3-, a product that may appear during the degradation process, was also detected. - -N and NO2 -The content of -N was tested. This included testing for NH4+. + The method used to measure NO3- content is Nessler's reagent spectrophotometry. - The method used to measure NO2 content is ultraviolet spectrophotometry. - The method used to determine the -N content is the N-(1-naphthyl)-ethylenediamine spectrophotometric method.

[0060] like Figure 2 As shown, at 36 h, this strain showed resistance to NH4+. + The removal efficiency of NO3- reached 99%, with an average removal rate of 2.78 mg / L / h from 0 to 36 h, and no NO3 was removed during this period. - -N, NO2 - Accumulation of -N.

[0061] When NH4Cl is used as the sole nitrogen source in the culture medium, the strain effectively reduces the concentration of ammonia nitrogen, and there is virtually no accumulation of nitrate and nitrite nitrogen.

[0062] Example 4:

[0063] Denitrification performance of Pseudomonas yinchengensis BK17 at low temperature (10℃) with nitrate nitrogen as the sole nitrogen source

[0064] The BK17 strain from Example 1 was inoculated into denitrification medium and then cultured in a shaker at 10°C. After it reached the logarithmic growth phase, 1% by volume of the culture solution was inoculated into 100 mL of NO3-. - The strain was cultured for 60 h in a nitrification medium with -N as the sole nitrogen source (concentration 100 mg / L) at 10 °C and 120 r / min. Samples were taken at 0, 12, 24, 36, 48, and 60 h, and the heterotrophic nitrification performance of the strain was tested after centrifugation and filtration. Simultaneously, NH4+, a product that may appear during the degradation process, was also detected. + -N and NO2 - The content of -N was tested. This included testing for NH4+. + The method used to measure NO3- content is Nessler's reagent spectrophotometry. - The method used to measure NO2 content is ultraviolet spectrophotometry. - The method used to determine the -N content is the N-(1-naphthyl)-ethylenediamine spectrophotometric method.

[0065] like Figure 3 As shown, the removal efficiency of this strain for NO3--N reached 75.68% at 42h, and NH4--N was reduced to 75.68% at 60h. + The removal efficiency of -N reached 83.64%, and the average removal rate from 0 to 242 h was 1.81 mg / L / h.

[0066] When KNO3 is used as the sole nitrogen source in the culture medium, this strain is resistant to NO3. - Although the degradation of -N did not affect NH4 + -N degrades rapidly, but can still effectively degrade NO3. - -N, and there is virtually no accumulation of ammonia nitrogen and nitrite nitrogen.

[0067] Example 5:

[0068] Denitrification performance of Pseudomonas yinchengensis BK17 at low temperature (10℃) using ammonia and nitrate nitrogen as a mixed nitrogen source

[0069] The BK17 strain from Example 1 was inoculated into denitrification medium and then cultured in a shaker at 10°C. After it reached the logarithmic growth phase, 1% by volume of the culture solution was inoculated into 100 mL of NH4+. + -N (concentration of 70 mg / L) and NO3 - The culture medium, using nitrification-denitrification (nitrification-denitrification) as the nitrogen source (30 mg / L), was incubated for 60 h at 10 °C and 120 rpm. Samples were taken at 0, 12, 24, 36, 48, and 60 h, and the NH4+ content in the supernatant was measured after centrifugation and filtration. + -N, NO3 - -N and NO2 - -N concentration was tested. + The method used to measure NO3- content is Nessler's reagent spectrophotometry. - The method used to measure NO2 content is ultraviolet spectrophotometry. - The method used to determine the -N content is the N-(1-naphthyl)-ethylenediamine spectrophotometric method.

[0070] like Figure 4 As shown, when ammonia nitrogen and nitrate nitrogen are present simultaneously, the strain preferentially utilizes ammonia nitrogen, and at 36 hours, the strain preferentially utilizes NH4+. + The removal efficiency of -N reached 99.51%, and NH4+ + -N was almost completely removed, at which point the strain began to degrade NO3. - -N, NO3 up to 60h - The removal efficiency of NO2- reached 99.12%, with almost no NO2 removal during the process. - -N accumulation.

[0071] When a mixed nitrogen source of NH4Cl and KNO3 is used as the culture medium, the strain preferentially utilizes ammonia nitrogen, and then utilizes nitrate nitrogen, with almost no accumulation of nitrite nitrogen during the degradation process.

[0072] Example 6:

[0073] Factors affecting heterotrophic nitrification of strain BK17:

[0074] The effect of carbon source on heterotrophic nitrification: The C / N ratio of the culture medium was fixed at 8. The carbon sources were adjusted to sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate. 1% of the seed culture was taken with a sterile pipette and placed in 100 ml of liquid culture medium with each of the above carbon sources. The culture was carried out at 10℃ and 120 rpm with shaking for 60 h. Samples were taken at 0, 12, 24, 36, 48, and 60 h. After centrifugation and filtration, the NH4+ in the supernatant was measured. + -N content and OD 600 The value was repeated in three parallel experiments, and the results were as follows: Figure 5 .

[0075] The effect of C / N ratio on heterotrophic nitrification: The C / N ratios in the culture medium were adjusted to 4, 8, 12, 16, and 20. 1% of the seed culture was taken using a sterile pipette and placed in 100 ml of the liquid culture medium at each of the different C / N ratios. The medium was incubated at 10℃ and 120 rpm with shaking for 60 h. Samples were taken at 0, 12, 24, 36, 48, and 60 h. After centrifugation and filtration, the NH4+ content in the supernatant was measured. + -N content and OD 600 The value was repeated in three parallel experiments, and the results were as follows: Figure 6 .

[0076] Effect of pH on heterotrophic nitrification: The pH of the culture medium was adjusted to 5, 6, 7, 8, and 9. 1% of the seed culture was taken using a sterile pipette and placed in 100 ml of liquid culture medium at each of the above different pH values. The medium was incubated at 10℃ and 120 rpm with shaking for 60 h. Samples were taken at 0, 12, 24, 36, 48, and 60 h. After centrifugation and filtration, the NH4+ content in the supernatant was measured. + -N content and OD 600 The value was repeated in three parallel experiments, and the results were as follows: Figure 7 .

[0077] Effect of rotation speed on heterotrophic nitrification: 1% of the seed culture was aspirated using a sterile pipette and placed in 100 ml of liquid culture medium. The culture was then incubated at 10°C with shaking at 40, 80, 120, 160, and 200 rpm for 60 h. Samples were taken at 0, 12, 24, 36, 48, and 60 h. After centrifugation and filtration, the NH4+ content in the supernatant was measured. + -N content and OD 600 The value was repeated in three parallel experiments, and the results were as follows: Figure 8 .

[0078] Effect of inoculum size on heterotrophic nitrification: 0.5%, 1%, 3%, 5%, and 7% of the seed culture were respectively pipetted and placed in 100 ml of liquid culture medium. The mixture was incubated at 10°C with shaking at 120 rpm for 60 h. Samples were taken at 0, 12, 24, 36, 48, and 60 h. After centrifugation and filtration, the NH4+ content in the supernatant was measured. + -N content and OD 600 The value was repeated in three parallel experiments, and the results were as follows: Figure 9 .

[0079] The results showed that ( Figures 5-9 The optimal carbon source for this strain is sodium citrate. The removal efficiency is best in the range of C / N ratio of 8-16, reaching up to 100%. The efficiency is best in the range of pH 6-8. The suitable rotation speed is 80-160 rpm, and the optimal inoculum size is 3%-5%.

[0080] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for wastewater denitrification using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria, characterized in that, The heterotrophic nitrifying aerobic denitrifying bacteria is *Pseudomonas cucurbita*, and its name is *Pseudomonas yinchengensis* (…). Pseudomonas umsongensis BK17, belonging to the genus Pseudomonas, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20242924 and deposit date of December 27, 2024. (1) Source of strain: The bacterial strains screened in the experiment were obtained from water samples collected from activated sludge in the aerobic tanks of wastewater treatment plants. (2) Culture medium formulation: Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L; Nitrification liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; Denitrification liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml; Nitrified solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L; Denitrification solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L; Mixed culture medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.2675 g / L, KNO3 0.2164 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; (3) Specific process: 1) Prepare enrichment medium. Add the sample to a 250ml Erlenmeyer flask containing 100mL of enrichment medium and incubate for 48h. Set the parameters of the constant temperature shaker to 10℃ and 120 r / min. When the cells grow to the point that the medium is obviously turbid, transfer 10mL of the cell suspension from the turbid medium to 100ml of fresh enrichment medium and continue to incubate at 10℃ and 120 r / min for 48h. Repeat the above process 3 times. 2) After enrichment, the samples were diluted using a gradient dilution method to obtain bacterial suspensions of 10⁻¹, 10⁻², ..., 10⁻⁸. Three gradients of 10⁻⁶, 10⁻⁷, and 10⁻⁸ were selected, and 100 μL of each gradient was evenly spread on heterotrophic nitrification solid medium. The suspensions were incubated at 10℃ for 2-3 days. Single colonies will form in the medium. Single colonies were picked and streaked multiple times in a three-zone heterotrophic nitrification solid medium to ensure the purity of the strains in the medium. 3) The obtained pure strains were inoculated onto denitrification medium and incubated in a biochemical incubator at 10°C for 72 h; strains with denitrification ability were preliminarily screened based on the color change of the denitrification medium, and strains that showed a blue halo were selected for further screening. 4) The selected strains were inoculated into nitrification liquid culture medium and cultured with shaking at 10℃ and 120 rpm. After 60 h, the ammonia nitrogen removal efficiency was measured, and the strain with the highest efficiency, BK17, was selected for subsequent experiments. (4) Application of wastewater denitrification methods: The process includes the following steps: Heterotrophic nitrifying aerobic denitrifying bacteria are cultured under different conditions at low temperatures, and their ammonia nitrogen removal efficiency is tested; the carbon sources for nitrogen-containing wastewater are sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate, with a C / N ratio of 4-20, a pH of 5-10, a rotation speed of 40-200 rpm, an inoculum size of 0.5%-4%, and a salinity of 0-4%.

2. The method for using low-temperature resistant heterotrophic nitrifying aerobic denitrifying bacteria for wastewater denitrification according to claim 1, characterized in that, The Pseudomonas BK17 strain removes one or a combination of ammonia nitrogen and nitrate nitrogen under low temperature conditions.

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